Abstract

RNA provides a stimulus for innate immunity either by activating the endosomal Toll-like receptors (TLRs) 3, 7 and 8 or by triggering cytosolic RNA sensors of the RIG-I-like family (RLRs) or the NALP3-containing inflammasome. RNA recognition results in the secretion of type-I interferons or proinflammatory cytokines. Recognition of RNA is supposed to occur due to the presence of non-self structures, such as the abundance of nucleotide modifications, guanosine-uridine-rich sequences, double-stranded conformation or the presence of a terminal 5’-triphosphate. However, the exact structural requirements for RNA recognition are still largely unknown. Within this work the functional and structural interplay between RNA and different receptors within cells of the innate immune system was analyzed. In detail, bacterial RNA, synthetic siRNA and in vitro transcribed RNA was characterized. It can be shown that TLR7 is the responsible receptor for the recognition of siRNA in plasmacytoid dendritic cells resulting in induction of type-I interferons which mediate unwanted off-target effects. Besides a minor sequence-dependent influence, introduction of nucleotide modifications identified the 2’-position of the ribose to be crucial for TLR7-mediated immunorecognition. Further, hyperchromicity assays and structure prediction give strong evidence that stimulating RNA adopts a duplex structure. These findings suggest that immunorecognition takes place within the minor groove of a given RNA duplex, thus revealing a possible mechanism of receptor ligand interaction. Confirming this notion, the introduction of different nucleotide modifications altering major groove recognition showed no effect on immunorecognition. Of note, incorporation of thymidine substitutions was demonstrated to reduce immunostimulation without any effect on gene knockdown. These results identify modifications that dissect unwanted immunostimulation from RNA interference in siRNA, thus paving the way for more precise therapeutic applications. Next, the immunostimulatory potential of different prokaryotic RNAs was tested. It could be shown that recognition of bacterial RNA differs from all principles described so far. Whereas TLR7 mediated recognition in plasmacytoid dendritic cells and NALP3 was responsible for IL-1β secretion, prokaryotic RNA was shown to additionally trigger a cytosolic receptor system resulting in the activation of the transcription factor NFκB and interleukin-12p40 secretion. Using cells from various knockout mice and performing siRNA approaches, all described RNA receptors could be ruled out to be responsible for this activation. Immunostimulation by prokaryotic RNA thus mirrors findings previously described for cytosolic DNA recognition. Finally, ligand specificity of RIG-I was investigated. It could be demonstrated that the 5’-triphosphate moiety is not the only target structure for RIG-I but that RNA in double-stranded conformation represents a second independent structural feature that activates RIG-I. The sensitivity of RIG-I to double-stranded RNA of increasing size was significantly enhanced, indicating that the biological role of RIG-I is rather the sensing of long dsRNA instead of recognizing the terminal 5’-end. Further, there is strong evidence that RIG-I might bind in a cooperative manner to double-stranded RNA structures explaining the significant length-dependency.